The present invention relates to a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide position, via partial digestion. The present invention further relates to a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide position via a combination of partial digestion and an oscillation reaction.
Genomic DNA provides the template for the information that allows the generation of proteins which are expressed and made by an organism. These proteins are generally essential for the survival of any specific cell in an organism. Therefore, the organism requires the template to be correct and free of mistakes in order to generate a protein that is functional in a cell. The protein may be nonfunctional if a single nucleotide of this DNA sequence is mutated (xe2x80x9ca point mutationxe2x80x9d). Point mutations which elicit disease states are known for many proteins.
Recent advances have allowed for the detection of point mutations with mismatch repair enzymes. Hsu et al., Carcinogenesis 15: 1657 (1994), describe the detection of A/G point mutations with mutY repair enzyme. Xu et al., Carcinogenesis 17(2): 321 (1996) further describe using mutY to detect A/G and to a lesser extent C/A mutations.
Youil et al., PNAS 92: 87 (1995) relate techniques for screening for each of the possible eight point mutations i.e. G/A, C/T, C/C, G/G, A/A, T/T, C/A, and G/T, using T4 endonuclease VII. Lu et al., WO93/20233 at 29-30 describe screening for mutations using all-type enzyme, which recognizes all eight base pair point mutations. Lu et al. also describe the use of combinations of different repair enzymes to ascertain the presence of an unknown point mutation in a sample. Id. at 27.
These techniques are directed, however, to the detection of a single point mutation in a given polynucleotide sample. To the extent that a nucleic acid target molecule has multiple base pair mutations spanning its length, the above methods are either ineffective or inefficient in detecting the presence and relative position of these mutations.
WO 96/40902 is also a background publication.
It is therefore an object of the present invention to provide a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide position via partial digestion.
It is a further object of the present invention to provide a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide position via a combination of partial digestion and an oscillation reaction.
In accomplishing the foregoing objects as well as other objects, there is provided a method of detecting the presence of and determining the relative positions of at least two point mutations in target polynucleotides, comprising:
(a) hybridizing single-stranded oligonucleotide probes to target polynucleotides to form hybrid, double-stranded polynucleotides such that mismatches occur at the sites of the point mutations, wherein the probes are complementary to a non-mutated sequence of the target polynucleotides and are labelled-at one end but not both ends, and wherein the target polynucleotides are not labelled;
(b) partially digesting the probe strands of the hybrid polynucleotides with a nucleic acid repair enzyme such that probe fragments of differing lengths are generated;
(c) separating the probe fragments by size in a medium suitable for visualizing the separated probe fragments; and then
(d) visualizing the separated probe fragments in the medium, whereby the presence and relative positions of the point mutations are determined.
There is further provided a method of detecting the presence of and determining the relative positions of at least two point mutations in a target polynucleotide, comprising:
(a) hybridizing a single-stranded oligonucleotide probe to a target polynucleotide to form a hybrid, double-stranded polynucleotide such that mismatches occur at the sites of the point mutations, wherein the probe is complementary to a non-mutated sequence of the target polynucleotide and is labelled at one end but not both ends, and wherein the target polynucleotide is not labelled;
(b) partially digesting the probe strand of the hybrid polynucleotide with a nucleic acid repair enzyme producing oligonucleotide fragments, wherein the oligonucleotide probe is designed such that the oligonucleotide fragments dissociate from the target polynucleotide spontaneously at a predetermined temperature;
(c) repeating steps (a) and (b) such that probe fragments of differing lengths are generated;
(d) separating the probe fragments by size in a medium suitable for visualizing the separated probe fragments; and then
(e) visualizing the separated probe fragments in the medium, whereby the presence and relative positions of the point mutations are determined.